The Regulation Of Immunostimulants On Immune System And The Prokaryotic Expression Of IL-10Gene On Chickens

In today’s poultry industry, immunosuppressive disease is an important risk in thedevelopment, such as infectious bursal disease virus (IBDV), Marek’s disease virus (MDV),chicken infectious anemia virus (CAV), avian leukosis virus (ALV), reticuloendothelialhyperplasia virus (REV), avian influenza virus (AIV), reovirus disease virus (REOV),hemorrhagic enteritis virus (HEV), adenovirus, cryptozoite, aspergillin, flavacin, fusariumbacteria, having brought serious economic losses to livestock breeding. And cytokines has avery important clinical significance and reference value in the prevention, control andtreatment process of immune suppression disease.In the first first part,60chickens,7-day-old,were randomly divided into four groups,as A1、A2、A3、A4,each group had15chickens,5chickens was a duplicate,there were three repeattotally.the A1is blank control group,bursal disease virus vaccination within21days of age inthe other three groups.A3group of chicks at25days to29days fed Shen’s immunostimulants1.2g/kg per chicken,A4group chicks injected ebony flavonoids at25days to29days0.2mlper chicken.At21days before challenged,25days,32days,46days,60days old, Bloodserum from SPF chicken wings vein was separated, the determination of serum IL-4, IL-10and TGF-beta and IFN-γ in each group were determination of four chickens randomly.In thesecond part,60chickens,7-day-old,were randomly divided into four groups,as B1、B2、B3、B4,each group had15chickens,5chickens was a duplicate,there were three repeat totally.In11days and25days of age,B2、B3and B4groups were inoculated with ND live vaccine,theB1is blank control group,bursal disease virus vaccination within21days of age in theB2group.B3group of chicks at25days to29days fed Shen’s immunostimulants1.2g/kg perchicken,B4group chicks injected ebony flavonoids at25days to29days0.2ml perchicken.At21days before challenged,25days,32days,46days,60days old, Blood serumfrom SPF chicken wings vein was separated, the determination of serum IL-4, IL-10andTGF-beta and IFN-γ in each group were determination of four chickens randomly. In thethird part, a pair of primers targeting IL-10gene in SPF Gallus gallus was designed according to the sequences of IL-10gene in the Chicken on GeneBank website. Total RNA fromPeripheral blood lymphocyte in SPF Gallus gallus breed was taken as template to makeRT-PCR amplification. IL-10gene in SPF Gallus gallus was cloned,sequenced andprokaryotic expression. The results showed that:The regulation of immunostimulants to SPF chickens immune suppression disease：Chickens infected with IBDV were in immunosuppression, the levels of IL-4, IL-10, IFN-γand TGF-β in the serum rose with varying degrees after infection;Shen’s immune enhancerand ebony flavonoids injection can immune suppression caused by IBDV with a good effect,and can effectivly prevent and treat the chicks immunosuppressive disease.The enhancementof immunostimulants on vaccine：The Shen immunostimulants and ebony flavonoids injectionHad some influence on the content of IL-4in chick serum;Shen’s immunostimulants andebony flavonoids injection inhibited the secretion of IL-10in chick serum;Shen’simmunostimulants and ebony flavonoids injection were not obviously affected the content ofIFN-γ and TGF-β in serum of chickens. Cloning and Distribution of interleukin-10Genes inSPF Gallus：We have cloned the IL-10gene successfully, with the DNAStar software analysisshowed that sequences are consistent with the chicken published in GenBank IL-10genesequences, indicating that we successfully cloned the IL-10gene in SPF chicken. Nucleotidehomology with other animals comparison shows that the identity is low, so it can beconsidered as a separate subgroup. According to bioinformatics analysis, IL-10gene encodesa protein with a hydrophobic and strong antigen, IL-10protein molecules contain at least8α-helices,12β-sheet. Chicken IL-10gene has six O-glycosylation sites, and noneN-glycosylation sites.PET-32a-IL-10prokaryotic expression recombinant plasmid wassuccessfully constructed, the right positive recombinant bacteria was induced by IPTG, forabout38.2ku expression products.